Our goal is to understand the mechanisms that underlie cellular differentiation. An example of this process is the asymmetric distribution of cellular components prior to division that leads to the genesis of progeny cells with different characteristics. This expression of asymmetry is a crucial event in normal development. A major landmark of asymmetry in Caulobacter is the localized biogenesis of a flagellum and the chemotaxis apparatus at one pole of the predivisional cell. The genes required to build the flagellum are ordered in a regulatory hierarchy that is initiated at a defined time in the cell cycle. The proposed research has three main objectives. The first is to identify the mechanisms responsible for the temporally controlled onset of expression of the flagellar regulatory hierarchy. To do this we will identify the promoter, regulatory sequences and trans-acting factors that control the temporal expression of the initial genes in the hierarchy, and isolate and characterize mutants that affect their expression or timing. In this context, we have shown that rpoN, encoding sigma54, is required for all polar differentiation events. The transcription of rpoN is temporally controlled and required for subsequent steps in the flagellar hierarchy. We will examine the control of rpoN expression and its global function. The second objective is based on our recent observations that chemotaxis proteins are localized at the cell pole in e. coli as well as in Caulobacter. We will determine if there are general mechanisms that lead to polar localization of the chemotaxis and flagella proteins and their subsequent proteolysis during morphogenesis. The third objective is to determine if the order of structural gene expression is essential for the assembly of the polar flagellum. It may be that the cell is composed of 'molecular machines', such as the chemotaxis apparatus, the cell division apparatus, and a polar complex used for flagellar assembly, that form spatially segregated functional groups of proteins. The bacterial cell appears, in fact, to be quite highly organized, and much is to be learned about the mechanisms of polarity, positional information, and targeting of proteins and protein complexes, in a relatively simple cell that is completely amenable to genetic and biochemical manipulation.